Increasing evidence suggests that the pH of the airway surface liquid (pHASL) plays an important role in inflammatory airway diseases, such as cystic fibrosis and asthma. Normal pHASL is slightly acidic (pH = 6.9) compared to plasma (pH = 7.4). The source of the acidity is not known. In our preliminary data, we show that human airway surface epithelia secrete acid by way of an apical membrane H+ Acid secretion is regulated by mucosal histamine and ATP, two mediators that are released in vivo during airway inflammation and stress. In studies on a variety of airway epithelial cell cultures, H+ secretion was only present in those cultures that expressed mRNA for the putative proton channel NADPH oxidase homologue 4 (NOX4). Therefore, based on these preliminary data we hypothesize that acidification of the airway surface liquid is caused by a NOX4 proton conductance. This hypothesis will be tested in three ways. In Specific Aim 1, we will determine the properties of the native H+ conductance and the underlying H+ channels expressed in airway surface epithelia using whole cell patch clamping, noise analysis, and pH stat recordings. The regulation of the H+ by the membrane voltage, intra- and extracellular pH, and its sensitivity to blockers will be studied. Physiological driving forces for H+ exit across the apical membrane will be measured using microelectrodes and confocal imaging. The connection between H+ secretion and superoxide generation by NADPH oxidase will be investigated. Specific Aim 2 is to determine the sequence and molecular expression of NOX4 in human airways. We will clone full-length NOX4 cDNA from human airway surface cells. Plasma membrane localization of NOX4 will be determined using a NOX4-GFP fusion protein. Cell type-specific expression of NOX4 will be determined by in situ hybridization of tracheal sections. Specific Aim 3 is to identify the function of NOX4 as the H+ channel in airway epithelia. The biophysical and physiological parameters of the recombinant NOX4 H+ conductance will be determined and quantitatively compared to the native H+ conductance. Antisense probes for NOX4 will be used to selectively inhibit expression of a H+ conductance in airway cells expressing endogenous NOX4. The results of our studies will reveal the molecular, physiological, and biophysical properties of the native H+ conductance and NOX4 in airways. In the long term, this research will improve our understanding of the mechanism responsible for acidification of the airway surface liquid. This will help develop novel therapeutical targets for the treatment of airway acidity and inflammatory airway diseases.